Hair cells are sensitive to a variety of environmental stresses, including aminoglycoside antibiotics, chemotherapeutic agents, and noise exposure, which are major causes of cell damage and hearing loss in the human population. Humans demonstrate variable susceptibiltiy to those stressors, yet little is known about the underlying molecular/genetic causes of this variability. Moreover, the biochemical mechanisms by which these factors lead to hair cell loss are poorly understood. Although the DNA damage pathway has been implicated in ototoxin-induced hair cell death, the DNA damage repair systems in hair and supporting cells remain largely uncharacterized. All organisms have evolved a number of distinct DNA repair systems to maintain the integrity of the genome;among them, nucleotide excision repair is a universal repair pathway involved in a wide spectrum of DNA-distorting lesions. Nucleotide excision repair is the only known molecular mechanism by which damage induced by chemotherapeutic agent cisplatin can be repaired, underlining its importance to hearing loss. In humans, inherited defects in the nucleotide excision repair pathway are implicated in Cockayne syndrome, a photosensitive disorder characterized by sensorineural hearing loss, premature aging, and severe neurological abnormalities caused by mutations in the CSA and CSB genes. Through a collaboration with Dr. Gijsbertus van der Horst (Erasmus University, Netherlands), we provide preliminary evidence that CSB-deficient mice manifest severe hearing loss in the high frequencies and outer hair cell degeneration by 16 weeks of age. We postulate that deficiency in nucleotide excision repair predisposes the outer hair cells to spontaneous, age-related loss. Testing the hypothesis that a normal, intrinsic attenuation of DNA repair pathways in hair cells is among the susceptibility factors in hearing loss is the main aim of this proposal. Our first objective is to characterize fully both the hearing deficits and inner ear degeneration in CSA- and CSB-deficient mice, through quantitative analysis of outer hair cell loss, apoptotic profiles and DNA damage response profiles. Our second objective is to characterize nucleotide excision repair in the normal organ of Corti using pharmacological and genetic approaches. The final objective is to test the hypothesis that CSA- and CSB-deficient mice are hypersensitive to ototoxic agents, both in organotypic cultures and in vivo. A thorough investigation of the DNA repair pathway in the inner ear is likely to provide new molecular targets for the possible treatment and prevention of hair cell death resulting from environmental toxic exposure.